Liposomes are widely used for drug delivery and for delivery of foreign DNA to mammalian cells, and they offer several advantages in this respect. Such advantages are ease of manufacture, commercial availability, the possibility of targeting to specific cells or tissues, and high transfection efficiency. Cationic liposomes, that is liposomes comprising at least some positively charged lipids to give an overall positive charge to the liposomes, have been used for gene transfer, i.e. transfection of DNA, into mammalian cells both in vivo and in vitro. Due to charge interactions, the positively charged liposome forms easily, in a simple mixing process, on its surface a complex with the negatively charged DNA. The complex in turn binds strongly to the cell surface due to favorable charge interactions followed by internalization of the complex into the cell and expression of the gene (Singhal, A. and Huang, L. (1994) Gene TheraPautics (ed.: Wolff, J. A.) pp 118-142, Birkhauser, Boston).
Various cationic lipids have been suggested in the art for incorporation into liposomes, including quaternary ammonium detergents, e.g, cetyltrimethylammonium bromide (CTAB), cationic derivatives of cholesterol and diacyl glycerol, e.g. 1,2-dioleyl-3-(4'-trimethylammonio) butanoyl-sn-glycerol (DOTB), and lipopolyamines, e.g. lipopoly-L-lysine (LPLL). Also commercial preparations are available. Lipofectin.RTM. "LIPOFECTIN" is a widely used commercial preparation comprising N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) in combination with dioleoylphosphatidylethanolamine (DOPE) (Gibco). The apparent toxicity of some of the commercially available products have been referred to the non-natural, non-biodegradable nature of compounds contained by them (Singhal et al, ibid).
The cationic liposomes used as a transfection vector commonly contain, in addition to the cationic lipid, a neutral or negatively charged lipid, so called helper lipid or co-lipid, the above mentioned DOPE being one such neutral helper lipid. Also other helper lipids have been suggested. Such helper lipids are typically phospholipids, and besides DOPE, e.g. dioleoylphosphatidylcholine (DOPC), dioleoylphatidylserine (DOPS), the corresponding dilauroyl, dimyristoyl and dipalmitoyl compounds, phosphatidic acid, phosphatidylglycerol, sterols, such as cholesterol, and mixtures of these have been suggested. The mono-, di- and triglycerides may be mentioned as further neutral helper lipids. A main function of the helper lipid is to fuse into and stabilize the liposome bilayer structure. It is also known that DOPE, in addition to the liposomal stabilizing effect, aids in the cytoplasmic delivery of DNA in the cell.
A wide variety of patent literature is available disclosing various cationic lipids for use for transfection, see e.g. U.S. Pat. No. 5,264,618, WO 93/03709, and WO 95/17373, just to mention a few.
Although a number of advantages are obtained by using cationic liposomes as carriers for DNA, rather than neutral or anionic liposomes, many problems still remain both in in vivo and in vitro applications. A major problem with many of the cationic lipids is that they are generally toxic to the cells, and thus of limited use. This is especially true of "LIPOFECTIN", the DOTMA component of which is a diether, and not-readily degraded in vivo and toxic to tissue cells. Thus DOTMA is not optimal from the point of view of in vivo gene delivery.
According to the invention it has now been discovered that by using a specific group of cationic lipids for inclusion into the transfection vector, it is possible to provide a method of transfection displaying clearly improved transfection efficiencies with minimal toxicity problems to the transfected cells.